The Interferons constitutes a family of proteins characterized by their non-specific antiviral and antiproliferative activity, a property that has made them useful as antiviral and anticancer drugs. Interferons are produced and released by animal cells upon exposure to a variety of inducing agents, the most potent of which are viruses. They are classified into three types: α-Interferon, β-Interferon, and γ-Interferon, based on serological and structural relations. The use of interferons as therapeutic drugs dates back to the 1970's. Although all three types of interferons have been subject to evaluation, α-interferon has become most widely used for therapeutic purposes. Among the interferons of human origin, the α-interferons are divided into several subtypes, which are either encoded by different gene loci or alleles of those, while there is only one subtype each of human β- and γ-interferon. The function of each subtype is still not clear, and the molecular or cellular targets of their antiviral and antineoplastic activities is thus not fully investigated. However, some in vitro studies have shown a larger biological effect when a mixture of different subtypes was used compared to using a single subtype only (Fan, S. X, Skillman, D. R, Liao, M-J, Testa, D. and Meltzer, M. S. (1993) AIDS Res. And Human Retrovir. 9, 1115–1122, Heim, A., Brehm, C., Stille-Siegener, M., Müller, G., Hake, S, Kandolf, R. and Figulla, H-R. (1995) J. Mol. Cell Cardiol. 27, 2199–2208).
There are presently three major methods for industry-scale production of α-interferon, all with fundamental differences in the cell system used. In the procaryotic systems, the gene coding for a single subtype, almost exclusively interferon α2, has been transferred to Escherichia coli, whereby this protein is expressed by the bacteria and subsequently harvested (Wessmann, C., Hagata, S., Boll, W., et al (1982) Phil. Trans. Royal Soc. London, Series B: Biol. Sci. 299 7–28). This process for the production of α-interferon is referred to herein as “recombinant interferon”. The bacterial cultures can be used for producing α-interferon at high efficiency, leading to an economically advantageous alternative to cells of human origin. However, only one single subtype is produced and the proteins are not modified (e.g. glycosylated) in contrast to interferon α2 that is produced in eukaryotic cells. A clinical drawback encountered with the recombinant α-interferon products is their tendency to induce antibodies against α-interferon in some patients. These neutralising antibodies have in several publications been shown to negatively affect the therapeutic treatment with recombinant α-interferon (Antonelli, G., Simeoni, E., Currenti, M., DePisa, F., Colizzi, V., Pistello, M., and Dianzani, F., (1997) Biother 10, 7–14, Öberg, K. and Alm, G. (1997) Biother 10, 1–5).
Alternatively α-interferon can be produced in human cells, either from established cell lines which are grown in vitro, or from primary cells, e.g., from peripheral leukocytes obtained as by-products from donated blood. In these case a mixture of α-interferon subtypes is obtained, although different cell sources produce a different subtype pattern. (Goren, T., Fischer, D. G. and Rubinstein M. (1986) J. Interferon Res. 6, 323–329. Established cell lines are clones derived from human tumors or from cells that have been immortalised, e.g., by the treatment with Epstein-Barr virus. These cells divide and grow indefinitely, in suitable media and under appropriate conditions. In contrast, interferon-producing primary cells such as leukocytes do not divide and have a finite life span. Such cells are consequently in limited supply, and their availability is a limiting factor for large scale production of native leukocyte α-interferon. Means for increasing the yield in the production process are therefore necessary.
Eucaryotic cell systems produce very little or no α-interferon spontaneously. The use of an “inducer” is therefore needed to initiate the production of α-interferon by the cells. Consequently, a large number of factors have been reported to initiate the production of α-interferon in various in vitro cell systems or in vivo. The most common inducers are different viruses, but synthetic organic substances have also been shown to act as inducers for the production of α-interferon. Some examples are leu-enkephalin and naloxone in vivo in mice (Gabrilovac, J., Ikic-Sutlic, M., Knezevic, N. and Poljak, L. (1996) Res. Exp. Med. 196, 137–144), neovir in vivo in mice (Mezentseva M., Narovlyansky, A., Kondratieva, T and Ershov, F. (1997). J. Interferon Res. 17, Suppl. 2, S94. Abstract), dipyridamole in vivo in humans (Galabov A. S. and Mastikova M. (1984) Biomed. Pharmacoth. 38, 412–413), 2-amino-5-bromo-6-methyl-4-pyrimidinol (2-ABMP) and its derivatives in vivo in humans(U.S. Pat. No. 3,932,617, Stringfellow, D. A., Vanderberg, H. C. and Weed, S. D. (1980) J. Interferon Res. 1, 1–14) and antraquinone derivatives in vivo in a variety of species (U.S. Pat. No. 4,027,021). Ethanol is reported to induce β-interferon production in Madin-Darby bovine kidney (MDBK) cells in the absence of other inducers (Chelbi-Alix, M. K. and Chousterman, S. (1992) J. Biol. Chem. 267, 1741–1745).
There are some examples where the production of α-interferon has been increased by ancillary reagents, termed herein as “enhancers”, i.e., compound(s) which is/are capable of increasing the production of α-interferon in cells activated by an inducing agent, but does not itself induce production of a α-interferon. Characteristic of an enhancer is that it can be added either before the agent that induces α-interferon, or after the induction has taken place. Substances that are reported as enhancers of interferon production in human cells are, e.g., the calmodulin inhibitor trifluoperazine used on fibroblasts (Lin, H-Y. and Thacore, H. R. (1990) J. Interferon Res. 10, 375–378), dexamethasone used as a stimulator on a cell line of lymphoblastoid origin (U.S. Pat. No. 4,266,024) and sodium butyrate, also used as a stimulant in a lymphoblastoid cell line (EP 0097 353, EP 0000520). Furthermore, it has been reported that the purine derivative theophylline acts as an enhancer and increases the yield of α-interferon from mouse Lpa cells induced by poly I:C (Zahorska, R., Korbecki, M., and Barciszewski, J. (1995) Arch. Immunol. Ther. Exp. 43, 43–46), and the use of some synthetic organic compounds, preferably tetramethylurea (TMU) (European Patent No 0 097 353), or dimethylsulfoxide (DMSO) (U.S. Pat. No. 4,266,024) has been shown to increase the α-interferon yield from a lymphoblastoid cell line treated with an inducer.
In the work leading to the present invention some of the substances which has been reported as inducers or enhancers when used in vivo or on fibroblasts and on a cell line of lymphoblastoid origin has been tested on human leukocytes, with the aim to increase the α-interferon production, both before or after induction with Sendai virus, but without any positive result on the yield of α-interferon. In some experiments even a decrease in α-interferon production from human leukocytes was observed when adding compounds known as enhancers in cell lines. For instance, pre-treatment with sodium butyrate has been described as a stimulant that leads to an increased yield of α-interferon in a lymphoblastoid cell line (European Patent No 0 097 353; European Patent No 0 000 520). In the present work it was found that pre-treatment of primary leukocytes with sodium butyrate according to a similar procedure led to a decrease in the yield of α-interferons, which points at fundamental differences between cell lines and primary cells as production systems for α-interferon proteins.
It is accordingly an object of the present invention to provide an improved process for production of α-interferon in human leukocytes induced by virus.